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  • C07C323/52—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
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  • C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
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  • C07C323/60—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
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  • C07C323/64—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and sulfur atoms, not being part of thio groups, bound to the same carbon skeleton
  • C07C323/65—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and sulfur atoms, not being part of thio groups, bound to the same carbon skeleton containing sulfur atoms of sulfone or sulfoxide groups bound to the carbon skeleton

Definitions

• This invention relates to new compounds, pharmaceutical compositions and diagnostic kits comprising such compounds, and methods of using such compounds for inhibiting NAALADase enzyme activity, detecting diseases where NAALADase levels
• NAALADase enzyme also known as prostate specific membrane antigen (“PSM” or “PS A”) and human glutamate carboxypeptidase II (“GCP II”) , catalyzes the hydrolysis of the neuropeptide N-acetyl-aspartyl-glutamate (“NAAG”) to N- acetyl-aspartate (“NAA”) and glutamate. Based upon amino acid sequence homology, NAALADase has been assigned to the 28 family of peptidases.
• PSM prostate specific membrane antigen
• GCP II human glutamate carboxypeptidase II
• NAALADase inhibitors may be effective in treating ischemia, spinal cord injury, demyelinating diseases, Parkinson's disease, Amyotrophic Lateral Sclerosis ("ALS"), alcohol dependence, nicotine dependence, cocaine dependence, cancer, diabetic neuropathy, pain and schizophrenia, and in inhibiting angiogenesis.
• ALS Amyotrophic Lateral Sclerosis
• This invention relates to a compound of formula I
• R 2 , R 3 , R 4 , R 5 , R s , R 7 and R 8 are independently hydrogen or C ⁇ -C 3 alkyl;
• a 1 , A 2 , A 3 and A 4 are independently hydrogen, C ⁇ -C 6 alkyl, C ⁇ -C 6 alkoxy, halo, nitro, phenyl, phenoxy, benzyl, benzyloxy or -COOH, or any adjacent two of A 2 , A 3 and A 4 form with the benzene ring a fused 5- or 6-membered carbocyclic or heterocyclic aromatic ring, said heterocyclic aromatic ring containing 1 or 2 oxygen, nitrogen and/or sulfur heteroatom(s) .
• This invention further relates to a compound of formula II
• This invention also relates to a compound of formula III
• X and Y are independently -CRR -, -0-, -S- or -NR provided that at least one of X and Y is/are - CR R - ;
• R, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, C ⁇ -C 9 alkyl, C 2 -C 9 alkenyl, C 2 -C 9 alkynyl, aryl, heteroaryl, carbocycle or heterocycle; and said alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle, heterocycle, alkoxy, alkenyloxy, phenoxy, benzyloxy, and fused ring are independently unsubstituted or substituted with one or more substituent (s) ; provided that if A 1 , A 2 and A 3 are each hydrogen, and A 4 and A 5 are each -COOH, then A 4 is ortho to A 5 ; and provided that if Y is -CR 5 R 6 -, then at least one of A 1 , A 2 , A 3 , A 4 and A 5 is/are independently phenoxy, benzyloxy,
• TGF- ⁇ activity comprising administering to a mammal in need of such inhibition, treatment or effect, an effective amount of a compound of formula I, II or III, as described above .
• This invention further relates to method for detecting a disease, disorder or condition where NAALADase levels are altered, comprising:
• This invention also relates to a method for detecting a disease, disorder or condition where NAALADase levels are altered in an animal or a mammal, comprising:
• this invention further relates to a diagnostic kit for detecting a disease, disorder or condition where NAALADase levels are altered, comprising a compound of formula I, II or III, as defined above, labeled with a marker.
• this invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising:
• FIG. 1 is a bar graph showing the effect of 2-
• Compound C (phosphonomethyl)pentanedioic acid
• FIG. 2 is a bar graph showing the effect of Compound C
• FIG. 3 is a bar graph showing the reversal of the
• FIG. 4 is a bar graph showing the non-reversal of the neuroprotective effect of Compound C by FGF neutralizing antibodies in ischemic cell cultures.
• FIG. 5 is a bar graph showing the reversal of the
• FIG. 6 is a bar graph showing the effect of Compound C
• FIG. 7A is a bar graph plotting the withdrawal latency difference scores of non-diabetic rats and STZ-diabetic rats treated with a vehicle or 2- [ [2, 3, 4, 5, 6- pentafluorobenzyl) hydroxyphosphinyl] methyl] entanedioic acid
• FIG. 7B is a bar graph plotting the withdrawal latency difference scores of non-diabetic rats and STZ-diabetic rats treated with a vehicle or 2- (2-sulfanylethyl) pentanedioic acid ("Compound D”) , against the days following administration with STZ.
• FIG. 8 is a bar graph plotting the withdrawal latency difference scores of normal (unoperated) rats and chronic constrictive injury-induced rats treated with a vehicle or Compound C, against the days following surgery.
• FIG. 9A is a bar graph plotting the motor nerve conduction velocity of non-diabetic rats and STZ-diabetic rats treated with a vehicle or Compound A, against the weeks post STZ.
• FIG. 9B is a bar graph plotting the sensory nerve conduction velocity of non-diabetic rats and STZ-diabetic rats treated with a vehicle or Compound A, against the weeks post STZ.
• FIG. 10A is a bar graph plotting the motor nerve conduction velocity of non-diabetic rats and STZ-diabetic rats treated with a vehicle or Compound D, against the weeks post STZ.
• FIG. 10B is a bar graph plotting the sensory nerve conduction velocity of non-diabetic rats and STZ-diabetic rats treated with a vehicle or Compound D, against the weeks post STZ.
• FIG. 11 is a graph plotting the withdrawal latency of non-diabetic rats and BB/W diabetic rats treated with a vehicle, Compound D or Compound A, against the weeks of treatment .
• FIG. 12 is a graph plotting the nerve conduction velocity of non-diabetic rats and BB/W diabetic rats treated with a vehicle, Compound D or Compound A, against the weeks of treatment .
• FIG. 13 is a bar graph plotting the withdrawal latency differences in scores of chronic constrictive injury-induced rats treated with a vehicle or 10 mg/kg 3-carboxy-5- (1, 1- dimethylethyl) -alpha- (3-mercaptopropyl) -benzenepropanoic acid ("Compound 49”), against the days following treatment.
• FIG. 14 is a bar graph plotting the withdrawal latency differences in scores of chronic constrictive injury-induced rats treated with a vehicle or 1 mg/kg Compound 49, against the days following treatment.
• FIG. 15 is a bar graph plotting the withdrawal latency differences in scores of chronic constrictive injury-induced rats treated with a vehicle or 0.1 mg/kg Compound 49, against the days following treatment.
• FIG. 16 is a bar graph plotting the withdrawal latency of chronic constrictive injury-induced rats treated with a vehicle or 10, 1 or 0.1 mg/kg Compound 49, against the days following treatment.
• FIG. 17 is a bar graph plotting the withdrawal latency differences in scores of chronic constrictive injury-induced rats treated with a vehicle or 50 mg/kg 3-carboxy-alpha- (3- mercaptopropyl) -benzenepropanoic acid ("Compound 5"), against the days following treatment.
• FIG. 18 is a bar graph plotting the withdrawal latency differences in scores of chronic constrictive injury-induced rats treated with a vehicle or 30 mg/kg Compound 5, against the days following treatment .
• FIG. 19 is a bar graph plotting the withdrawal latency differences in scores of chronic constrictive injury-induced rats treated with a vehicle or 10 mg/kg Compound 5, against the days following treatment.
• FIG. 20 is a bar graph plotting the withdrawal latency differences in scores of chronic constrictive injury-induced rats treated with a vehicle or 3 mg/kg Compound 5, against the days following treatment.
• FIG. 21 is a bar graph plotting the withdrawal latency differences in scores of chronic constrictive injury-induced rats treated with a vehicle or 1 mg/kg Compound 5, against the days following treatment.
• FIG. 22 is a bar graph plotting the withdrawal latency differences in scores of chronic constrictive injury-induced rats treated with a vehicle or 0.3 mg/kg Compound 5, against the days following treatment.
• FIG. 23 is a bar graph plotting the withdrawal latency of chronic constrictive injury-induced rats treated with a vehicle or 10, 30 or 50 mg/kg Compound 5, against the days following treatment.
• FIG. 24 is a bar graph plotting the percent of transgenic mice at 210 days of age that exhibited limb shaking after treatment with 2- (3- sulfanylpropyl) pentanedioic acid ("Compound B”) or a vehicle .
• FIG. 25 is a bar graph plotting the gait, measured
• FIG. 26 is a bar graph plotting hind limbs dragging
• transgenic mice at 210 days of age after treatment with
• FIG. 27 is a bar graph plotting the crossing of
• FIG. 28 is a bar graph plotting the righting reflex
• FIG. 29 is a graph plotting the percent of
• mice that died against the age of the mice (days) .
• FIG. 30 is a Kaplan-Meier survival graph plotting
• FIG. 31 is a bar graph plotting the withdrawal latency difference scores of non-diabetic rats and STZ-diabetic rats treated with a vehicle, Compound D or 3-carboxy-alpha- (3- mercaptopropyl)benzenepropanoic acid ("Compound E” ) , against the weeks of treatment .
• FIG. 32 is a bar graph plotting motor nerve conduction velocity of non-diabetic rats and STZ-diabetic rats treated with a vehicle, Compound D or Compound E, against the weeks of treatment .
• FIG. 33 is a bar graph plotting sensory nerve conduction velocity of non-diabetic rats and STZ-diabetic rats treated with a vehicle, Compound D or Compound E, against the weeks of treatment, where treatment started 5 weeks post STZ.
• FIG 34 is a bar graph plotting the withdrawal latency difference scores of non-diabetic rats and STZ-diabetic rats treated with a vehicle or lower doses of Compound D (1 and 3 mg/kg) , against the weeks of treatment, where treatment started 7 weeks post STZ .
• FIG. 35 is a bar graph plotting motor nerve conduction velocity of non-diabetic rats and STZ-diabetic rats treated with a vehicle or lower doses of Compound D (1 and 3 mg/kg) , against the weeks of treatment, where treatment started 7 weeks post STZ.
• FIG. 36 is a bar graph plotting sensory nerve conduction velocity of non-diabetic rats and STZ-diabetic rats treated with a vehicle or lower doses of Compound D (1 and 3 mg/kg) , against the weeks of treatment, where treatment started 7 weeks post STZ.
• FIG. 37 are bar graphs plotting sensory nerve conduction velocity of non-diabetic rats and STZ-diabetic rats treated with a vehicle or Compound D at 35 days and 60 days after treatment, where treatment started 60 days post
• FIG. 38 are bar graphs plotting motor nerve conduction velocity of non-diabetic rats and STZ-diabetic rats treated with a vehicle or Compound D at 35 days after treatment, where treatment started 60 days post STZ.
• FIG. 39 is a graph plotting sensory nerve conduction velocity of non-diabetic and STZ-diabetic rats treated with a vehicle or Compound D, against the days post STZ, where treatment started 90 days post STZ.
• FIG. 40 is a bar graph plotting motor and sensory nerve conduction velocities of non-diabetic mice and db/db diabetic mice before treatment with a NAALADase inhibitor.
• FIG. 41 is a bar graph plotting motor and sensory nerve conduction velocities of non-diabetic mice and db/db diabetic mice after treatment with 3-carboxy-5- (1, 1- dimethylethyl) -alpha- (3-mercaptopropyl)benzenepropanoic acid
• FIG. 42 is bar graph comparing the rotarod
• mice normal non-HD mice treated with a vehicle.
• FIG. 43 is a bar graph comparing the total distance
• mice normal non-HD mice treated with a vehicle.
• FIG. 44 is a graph plotting the survival time of
• transgenic D mice treated with Compound B or a vehicle.
• FIG. 45 is a graph plotting the survival time of
• FIG. 46 is a graph plotting the survival time of
• Compound A refers to 2- [ [2,3,4,5, 6- pentafluorobenzyl) hydroxyphosphinyl] methyl] pentanedioic acid.
• Compound B refers to 2- (3-sulfanylpropyl) pentanedioic acid.
• Compound C refers to 2- (phosphonomethyl) pentanedioic acid (PMPA) .
• Compound D refers to 2- (2-sulfanylethyl) pentanedioic acid.
• Compound E refers to 3-carboxy-alpha- (3- mercaptopropyl) benzenepropanoic acid.
• Compound F refers to 3 ⁇ carboxy-5- (1, 1-dimethylethyl) - alpha- (3-mercaptopropyl) benzenepropanoic acid.
• Alkyl refers to a branched or unbranched saturated hydrocarbon chain comprising a designated number of carbon atoms.
• C ⁇ -C 9 alkyl is a straight or branched hydrocarbon chain containing 1 to 9 carbon atoms, and includes but is not limited to substituents such as methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl, and the like, unless otherwise indicated.
• alkenyl refers to a branched or unbranched unsaturated hydrocarbon chain comprising a designated number of carbon atoms.
• C 2 -C 9 alkenyl is a straight or branched hydrocarbon chain containing 2 to 9 carbon atoms having at least one double bond, and includes but is not limited to substituents such as ethenyl, propenyl, iso- propenyl, butenyl, iso-butenyl, tert-butenyl, n-pentenyl, n- hexenyl, and the like, unless otherwise indicated.
• Alkoxy refers to the group -OR wherein R is alkyl as herein defined.
• R is a branched or unbranched saturated hydrocarbon chain containing 1 to 9 carbon atoms .
• Carbocycle refers to a hydrocarbon, cyclic moiety having one or more closed ring(s) that is/are alicyclic, aromatic, fused and/or bridged. Examples include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclopentene, cyclohexene, cycloheptene, cycloctene, benzyl, naphthene, anthracene, phenanthracene, biphenyl and pyrene.
• Aryl refers to an aromatic, hydrocarbon cyclic moiety having one or more closed rings. Examples include, without limitation, phenyl, benzyl, naphthyl, anthracenyl, phenanthracenyl , biphenyl and pyrenyl .
• Heterocycle refers to a cyclic moiety having one or more closed rings that is/are alicyclic, aromatic, fused and/or bridged, with one or more heteroatoms (for example, sulfur, nitrogen or oxygen) in at least one of the rings. Examples include, without limitation, pyrrolidine, pyrrole, thiazole, thiophene, piperidine, pyridine, isoxazolidine and isoxazole .
• Heteroaryl refers to an aromatic, cyclic moiety having one or more closed rings with one or more heteroatoms (for example, sulfur, nitrogen or oxygen) in at least one of the rings. Examples include, without limitation, pyrrole, thiophene, pyridine and isoxazole.
• Derivative refers to a substance produced from another substance either directly or by modification or partial substitution.
• Effective amount refers to the amount required to produce the desired effect, for example, to inhibit NAALADase enzyme activity and/or angiogenesis, to effect
• neuronal activity or TGF- ⁇ activity and/or to treat
• glutamate abnormality is glutamate abnormality, compulsive disorder, prostate disease, cancer or glaucoma.
• Electromagnetic radiation includes without limitation radiation having the wavelength of 10 "20 to 10° meters. Examples include, without limitation, gamma radiation (10 ⁇ 20 to 10 "13 m) , X-ray radiation (10 "11 to 10 "9 m) , ultraviolet light (10 nm to 400 nm) , visible light (400 nm to 700 nm) , infrared radiation (700 nm to 1.0 mm) and microwave radiation (1 mm to 30 cm) .
• Halo refers to at least one fluoro, chloro, bromo or iodo moiety.
• “Isosteres” refer to elements, functional groups, substitutents, molecules or ions having different molecular formulae but exhibiting similar or identical physical properties.
• tetrazole is an isostere of carboxylic acid because it mimics the properties of carboxylic acid even though they both have different molecular formulae.
• two isosteric molecules have similar or identical volumes and shapes.
• isosteric compounds should be isomorphic and able to co-crystallize.
• Other physical properties that isosteric compounds usually share include boiling point, density, viscosity and thermal conductivity. However, certain properties are usually different: dipolar moments, polarity, polarization, size and shape since the external orbitals may be hybridized differently.
• the term “isosteres” encompasses "bioisosteres” .
• Bioisosteres are isosteres that, in addition to their physical similarities, share some common biological properties. Typically, bioisosteres interact with the same recognition site or produce broadly similar biological effects.
• Carboxylic acid isosteres include without limitation direct derivatives such as hydroxamic acids, acyl-cyanamides and acylsuifonamides; planar acidic heterocycles such as tetrazoles, mercaptoazoles, sulfinylazoles, sulfonylazoles,
• NAALADase has been assigned to the 28 peptidase family and is also called PSMA or human GCP II, EC number 3.4.17.21. It is believed that NAALADase is a co-catalytic zinc/zinc metallopeptidase. NAALADase shows a high affinity for NAAG with . a Km of 540 nM. If NAAG is a bioactive peptide, then NAALADase may serve to inactivate NAAG'S synaptic action. Alternatively, if NAAG functions as a precursor for glutamate, the primary function of NAALADase may be to regulate synaptic glutamate availability.
• “Pharmaceutically acceptable carrier” refers to any carrier, diluent, excipient, wetting agent, buffering agent, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavorant, or sweetener, preferably non-toxic, that would be suitable for use in a pharmaceutical composition.
• “Pharmaceutically acceptable equivalent” includes, without limitation, pharmaceutically acceptable salts, hydrates, metabolites, prodrugs and isosteres. Many pharmaceutically acceptable equivalents are expected to have the same or similar in vi tro or in vivo activity as the compounds of the invention.
• “Pharmaceutically acceptable salt” refers to a salt of the inventive compounds which possesses the desired pharmacological activity and which is neither biologically nor otherwise undesirable.
• the salt can be formed with acids that include, without limitation, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycero- phosphate, hemisulfate, heptanoate, hexanoate, hydrochloride hydrobromide, hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
• Examples of a base salt include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D- glucamine, and salts with amino acids such as arginine and lysine.
• Basic nitrogen-containing groups can be quarternized with agents including lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aralkyl halides such as benzyl and phenethyl bromides .
• lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides
• dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates
• long chain halides such as decyl, lauryl, myristyl and stearyl chlorides,
• Prodrug refers to a derivative of the inventive compounds that undergoes biotransformation, such as metabolism, before exhibiting its pharmacological effect (s).
• the prodrug is formulated with the objective (s) of improved chemical stability, improved patient acceptance and compliance, improved bioavailability, prolonged duration of action, improved organ selectivity, improved formulation (e.g., increased hydrosolubility) , and/or decreased side effects (e.g., toxicity).
• the prodrug can be readily prepared from the inventive compounds using methods known in the art, such as those described by Burger ' s Medicinal Chemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp. 172- 178, 949-982 (1995) .
• Radiosensitizer refers to a low molecular weight compound administered to animals in therapeutically effective amounts to promote the treatment of diseases that are treatable with electromagnetic radiation.
• Diseases that are treatable with electromagnetic radiation include, without limitation, neoplastic diseases, benign and malignant tumors, and cancerous cells. Electromagnetic radiation treatment of other diseases not listed herein are also contemplated by this invention.
• “Inhibition,” in the context of enzymes refers to reversible enzyme inhibition such as competitive, uncompetitive and non-competitive inhibition.
• Competitive, uncompetitive and non-competitive inhibition can be distinguished by the effects of an inhibitor on the reaction kinetics of an enzyme.
• Competitive inhibition occurs when the inhibitor combines reversibly with the enzyme in such a way that it competes with a normal substrate for binding at the active site. The affinity between the inhibitor and the enzyme may be measured by the inhibitor constant, Ki, which is defined as:
• Ki refers to the affinity between the inventive compounds and NAALADase.
• IC 50 is a related term used to define the concentration or amount of a compound that is required to cause a 50% inhibition of the target enzyme .
• NAALADase inhibitor refers to any compound that inhibits NAALADase enzyme activity.
• a NAALADase enzyme activity Preferably, a NAALADase
• inhibitor exhibits a Ki of less than 100 ⁇ M, more preferably
• “Isomers” refer to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the arrangement or configuration of the atoms .
• Optical isomers refer to enantiomers or diastereoisomers .
• Steps are isomers that differ only in the arrangement of the atoms in space .
• Diastereoisomers are stereoisomers that are not mirror images of each other. Diastereoisomers occur in compounds having two or more asymmetric carbon atoms; thus, such compounds have 2 n optical isomers, where n is the number of asymmetric carbon atoms.
• Enantiomers are a pair of stereoisomers that are non- superimposable mirror images of each other. Enantiomers result from the presence of one or more asymmetric carbon atoms in the compound (e.g., glyceraldehyde, lactic acid, sugars, tartaric acid, amino acids) . “Enantiomer-enriched” refers to a mixture in which one enantiomer predominates.
• Racemic mixture means a mixture containing equal amounts of individual enantiomers.
• Non-racemic mixture is a mixture containing unequal amounts of enantiomers.
• Angiogenesis refers to the process whereby new capillaries are formed.
• “Inhibition” of angiogenesis may be measured by many parameters in accordance with this invention and, for instance, may be assessed by delayed appearance of neovascular structures, slowed development of neovascular structures, decreased occurrence of neovascular structures, slowed or decreased severity of angiogenesis- dependent disease effects, arrested angiogenic growth, or regression of previous angiogenic growth. In the extreme, complete inhibition is referred to herein as prevention.
• prevention refers to no substantial angiogenesis or angiogenic growth if none had previously occurred, or no substantial further angiogenesis or angiogenic growth if growth had previously, occurred.
• Angiogenesis-dependent disease includes, without limitation, rheumatoid arthritis, cardiovascular diseases, neovascular diseases of the eye, peripheral vascular disorders, dermatologic ulcers and cancerous tumor growth, invasion and metastasis.
• Animal refers to a living organism having sensation and the power of voluntary movement, and which requires for its existence oxygen and organic food. Examples include, without limitation, members of the human, equine, porcine, bovine, murine, canine, or feline species. In the case of a human, an "animal” may also be referred to as a "patient” .
• “Mammal” refers to a warm-blooded vertebrate animal.
• “Anxiety” includes without limitation the unpleasant emotion state consisting of psychophysiological responses to anticipation of unreal or imagined danger, ostensibly resulting from unrecognized intrapsychic conflict.
• Physiological concomitants include increased heart rate, altered respiration rate, sweating, trembling, weakness, and fatigue; psychological concomitants include feelings of impending danger, powerlessness, apprehension, and tension.
• Physiological concomitants include increased heart rate, altered respiration rate, sweating, trembling, weakness, and fatigue; psychological concomitants include feelings of impending danger, powerlessness, apprehension, and tension.
• Dorland's Illustrated Medical Dictionary, .B. Saunders Co., 27th ed. (1988) “Anxiety Disorder” includes without limitation mental disorders in which anxiety and avoidance behavior predominate. Dorland's Illustrated Medical Dictionary, W.B. Saunders Co., 27th ed. (1988).
• Examples include without limitation panic attack, agoraphobia, panic disorder, acute stress disorder, chronic stress disorder, specific phobia, simple phobia, social phobia, substance induced anxiety disorder, organic anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, generalized anxiety disorder, and anxiety disorder NOS.
• Other anxiety disorders are characterized in Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association 4th ed. 1994) .
• Attention Deficit Disorder or "ADD” refers to a disorder characterized by developmentally inappropriate inattention and impulsiveness, with or without hyperactivity.
• Inattention means a failure to finish tasks started, easily distracted, seeming lack of attention, and difficulty concentrating on tasks requiring sustained attention.
• Impulsiveness means acting before thinking, difficulty taking turns, problems organizing work, and constant shifting from one activity to another. Hyperactivity means difficulty staying seated and sitting still, and running or climbing excessively.
• “Cancer” includes, without limitation, ACTH-producing tumors, acute lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer, brain cancer, breast cancer, cervix cancer, chronic lymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and/or non-small cell) , malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin ' s lymphoma, osteosarcoma, ovary cancer, ovary (ger
• Compulsive disorder refers to any disorder characterized by irresistible impulsive behavior. Examples of compulsive disorders include without limitation substance dependence, eating disorders, pathological gambling, Attention Deficit Disorder ("ADD”), and Tourette ' s syndrome.
• ADD Attention Deficit Disorder
• Demyelinating disease refers to any disease involving damage to or removal of the myelin sheath naturally surrounding nerve tissue, such as that defined in U.S. Patent No. 5,859,046 and International Publication No. WO 98/03178, herein incorporated by reference. Examples include without limitation peripheral demyelinating diseases (such as Guillain-Barre syndrome, peripheral neuropathies and Charcot-Marie Tooth disease) and central demyelinating diseases (such as multiple sclerosis) .
• peripheral demyelinating diseases such as Guillain-Barre syndrome, peripheral neuropathies and Charcot-Marie Tooth disease
• central demyelinating diseases such as multiple sclerosis
• Disease refers to any deviation from or interruption of the normal structure or function of any part, organ or system (or combinations) of the body that is manifested by a characteristic set of symptoms and signs and whose etiology, pathology, and prognosis may be known or unknown. Dorland' s Illustrated Medical Dictionary, (W.B. Saunders Co. 27th ed. 1988) .
• Disorder refers to any derangement or abnormality of function; a morbid physical or mental state. Dorland' s Illustrated Medical Dictionary, (W.B. Saunders Co. 27th ed. 1988) .
• Drug dependence refers to a psychologic addiction or a physical tolerance to a drug. Tolerance means a need to increase the dose progressively in order to produce the effect originally achieved by smaller amounts.
• Eating disorder refers to compulsive overeating, obesity or severe obesity.
• Obesity means body weight of 20% over standard height-weight tables.
• Severe obesity means over 100% overweight.
• Glaucoma includes without limitation chronic
• idiopathic open-angle glaucomas e.g., high-pressure, normal-pressure
• pupillary block glaucomas e.g., acute angle-closure, subacute angle-closure, chronic angle-closure, combined-mechanism
• developmental glaucomas e.g., congenital (infantile) , juvenile, Anxenfeld-Rieger syndrome, Peters' anomaly, Aniridia
• glaucomas associated with other ocular disorders e.g., glaucomas associated with disorders of the corneal endothelium, iris, ciliary body, lens, retina, choroid and vitreous
• glaucomas associated with elevated episcleral venous pressure e.g., systemic diseases with associated elevated intraocular pressure and glaucoma, corticosteroid-induced glaucoma
• glaucomas associated with inflammation and trauma e.g., glaucomas
• glutamate abnormality refers to any disease, disorder, or condition in which glutamate is implicated, including pathological conditions involving elevated levels of glutamate.
• glutamate abnormalities include, without limitation, compulsive disorder, spinal cord injury, epilepsy, stroke, ischemia, demyelinating disease, Alzheimer's disease, Parkinson's disease, ALS, Huntington's disease, schizophrenia, pain, peripheral neuropathy (including but not limited to diabetic neuropathy) , traumatic brain injury, neuronal insult, inflammatory disease, anxiety, anxiety disorder, memory impairment and glaucoma.
• Ischemia refers to localized tissue anemia due to obstruction of the inflow of arterial blood.
• Global ischemia occurs when blood flow ceases for a period of time, as may result from cardiac arrest.
• Focal ischemia occurs when a portion of the body, such as the brain, is deprived of its normal blood supply, such as may result from thromboembolytic occlusion of a cerebral vessel, traumatic head injury, edema or brain tumor. Even if transient, both global and focal ischemia can produce widespread neuronal damage. Although nerve tissue damage occurs over hours or even days following the onset of ischemia, some permanent nerve tissue damage may develop in the initial minutes following cessation of blood flow to the brain.
• Memory impairment refers to a diminished mental registration, retention or recall of past experiences, knowledge, ideas, sensations, thoughts or impressions.
• Memory impairment may affect short and long-term information retention, facility with spatial relationships, memory
• memory impairment is a common feature of neurodegenerative diseases such as Alzheimer's disease and senile dementia of the Alzheimer type. Memory impairment also occurs with other kinds of dementia such as multi- infarct dementia, a senile dementia caused by cerebrovascular deficiency, and the Lewy-body variant of Alzheimer's disease with or without association with Parkinson's disease. Creutzfeldt-Jakob disease is a rare dementia with which memory impairment is associated.
• Brain damage may occur, for example, after a classical stroke or as a result of an anaesthetic accident, head trauma, hypoglycemia, carbon monoxide poisoning, lithium intoxication, vitamin (Bi, thiamine and B ⁇ 2 ) deficiency, or excessive alcohol use.
• Korsakoff's amnesic psychosis is a rare disorder characterized by profound memory loss and confabulation, whereby the patient invents stories to conceal his or her memory loss. It is frequently associated with excessive alcohol intake.
• Memory impairment may furthermore be age-associated; the ability to recall information such as names, places and words seems to decrease with increasing age.
• Transient memory loss may also occur in patients, suffering from a major depressive disorder, after electro-convulsive therapy.
• Major depressive disorder refers to any clinically significant behavioral or psychological syndrome characterized by the presence of distressing symptoms or significant impairment of functioning. Mental disorders are assumed to result from some psychological or organic dysfunction of the individual; the concept does not include disturbances that are essentially conflicts between the individual and society (social deviance) .
• Methodastasis refers to "[t]he ability of cells of a cancer to disseminate and form new foci of growth at noncontiguous sites (i.e., to form metastases) .
• the transition from in si tu tumor growth to metastatic disease is defined by the ability of tumor cells of the primary site to invade local tissues and to cross tissue barriers ... To initiate the metastatic process, carcinoma cells must first penetrate the epithelial basement membrane and then invade the interstitial stroma.
• Neuro insult refers to any damage to nervous tissue and any disability or death resulting therefrom.
• the cause of nervous insult may be metabolic, toxic, neurotoxic, iatrogenic, thermal or chemical, and includes without limitation ischemia, hypoxia, cerebrovascular accident, trauma, surgery, pressure, mass effect, hemorrhage, radiation, vasospasm, neurodegenerative disease, neurodegenerative process, infection, Parkinson's disease, ALS, myelination/demyelination processes, epilepsy, cognitive disorder, glutamate abnormality and secondary effects thereof.
• Neuronal tissue refers to the various components that make up the nervous system, including without limitation neurons, neural support cells, glia, Schwann cells, vasculature contained within and supplying these structures, the central nervous system, the brain, the brain stem, the spinal cord, the junction of the central nervous system with the peripheral nervous system, the peripheral nervous system and allied structures.
• Neuroopathy refers to any disease or malfunction
• Neuropathy includes, without limitation,
• peripheral neuropathy diabetic neuropathy, autonomic
• diseases for example, amyloidosis, alcoholism, HIV,
• diabetes including type I and type II
• dapsone vitamins, paclitaxel chemotherapy, HAART
• nerve plexus for example, trauma, compression,
• Neuroprotective refers to the effect of reducing, arresting or ameliorating nervous insult, and protecting, resuscitating or reviving nervous tissue that has suffered nervous insult.
• Pain refers to localized sensations of discomfort, distress or agony, resulting from the stimulation of specialized nerve endings. It serves as a protective mechanism insofar as it induces the sufferer to remove or withdraw from the source. Dorland's Illustrated Medical Dictionary, (W.B. Saunders Co. 27th ed. 1988) .
• Examples of pain include, without limitation, acute, chronic, cancer, burn, incisional, inflammatory, neuropathic and back pain.
• Neuroopathic pain refers to a condition of pain associated with a nerve injury. Depending on the particular syndrome, the pain may be due to alterations of the brain or spinal cord or may be due to abnormalities in the nerve itself.
• Neuropathic pain may be idiopathic or induced by any causes including diseases (for example, amyloidosis, alcoholism, HIV, syphilis, virus, autoimmune disorder, cancer, porphyria, arachnoiditis, post herpetic neuralgia, Guillain-Barre syndrome, diabetes including type I and type II diabetes) , chemicals (for example, toxins, lead, dapsone, vitamins, paclitaxel chemotherapy, HAART therapy) and physical injuries to a particular nerve or nerve plexus (for example, trauma, compression, constriction) .
• diseases for example, amyloidosis, alcoholism, HIV, syphilis, virus, autoimmune disorder, cancer, porphyria, arachnoiditis, post herpetic neuralgia, Guillain-Barre syndrome, diabetes including type I and type II diabetes
• chemicals for example, toxins, lead, dapsone, vitamins, paclitaxel chemotherapy, HAART therapy
• prostate disease refers to any disease affecting the prostate. Examples of prostate disease include without limitation prostate cancer such as adenocarcinoma and metastatic cancers of the prostate; and conditions characterized by abnormal growth of prostatic epithelial cells such as benign prostatic hyperplasia.
• “Schizophrenia” refers to a mental disorder or group of mental disorders characterized by disturbances in form and content of thought (loosening of associations, delusions, hallucinations) , mood (blunted, flattened, inappropriate affect), sense of self and relationship to the external world (loss of ego boundaries, dereistic thinking, and autistic withdrawal) , and behavior (bizarre, apparently purposeless, and stereotyped activity or inactivity) .
• schizophrenia examples include, without limitation, acute, ambulatory, borderline, catatonic, childhood, disorganized, hebephrenic, latent, nuclear, paranoid, paraphrenic, prepsychotic, process, pseudoneurotic, pseudopsychopathic, reactive, residual, schizo-af eetive and undifferentiated schizophrenia. Dorland's Illustrated Medical Dictionary, (W.B. Saunders Co. 27th ed. 1988) .
• TGF- ⁇ refers to transforming growth factor beta.
• TGF- ⁇ is recognized as a prototype of multifunctional growth factors. It regulates various cell and tissue functions, including cell growth and differentiation, angiogenesis, wound healing, immune function, extracellular matrix production, cell chemotaxis, apoptosis and hematopoiesis .
• TGF- ⁇ abnormality refers to any disease, disorder or
• TGF- ⁇ condition in which TGF- ⁇ is implicated, including diseases disorders and conditions characterized by an abnormal level
• Therapeutic window of opportunity refers, in relation to stroke, to the maximal delay between the onset of stroke and the initiation of efficacious therapy.
• Tourette's syndrome refers to an autosomal multiple tic disorder characterized by compulsive swearing, multiple muscle tics and loud noises. Tics are brief, rapid, involuntary movements that can be simple or complex; they are stereotyped and repetitive, but not rhythmic. Simple tics, such as eye blinking, often begin as nervous mannerisms. Complex tics often resemble fragments of normal behavior.
• treating refers to:
• Treating ALS refers to:
• Substances for which dependence may be developed include, without limitation, depressants (opioids,
• Relapse refers to a return to substance use after
• “Tolerance” refers to an acquired reaction to a substance characterized by diminished effect with continued use of the same dose and/or a need for increased doses to achieve intoxication or desired effect previously achieved by lower doses. Both physiological and psychosocial factors may contribute to the development of tolerance. With respect to physiological tolerance, metabolic and/or functional tolerance may develop. By increasing the rate of metabolism of the substance, the body may be able to eliminate the substance more readily. Functional tolerance is defined as a decrease in sensitivity of the central nervous system to the substance .
• This invention relates to compounds of formula I
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen or C 1 -C3 alkyl
• a 1 , A 2 , A 3 and A 4 are independently hydrogen, O . -C 6 alkyl, C 1 -C6 alkoxy, halo, nitro, phenyl, phenoxy, benzyl, benzyloxy or -COOH, or any adjacent two of A 2 , A 3 and A 4 form with the benzene ring a fused 5- or 6-membered carbocyclic or heterocyclic aromatic ring, said heterocyclic aromatic ring containing 1 or 2 oxygen, nitrogen and/or sulfur heteroatom(s) .
• R 1 , R 2 , R 3 , R 4 , R ⁇ , R 6 , R 7 and R 8 are independently hydrogen or methyl; and A 1 , A 2 , A 3 and A 4 are independently hydrogen, C x -C alkyl, C ⁇ -C 2 alkoxy, halo, nitro, phenyl, phenoxy, benzyloxy, nitro or -COOH.
• any adjacent two of A 2 , A 3 and A 4 form with the benzene ring a fused 5- or 6-membered carbocyclic or heterocyclic aromatic ring, said heterocyclic aromatic ring containing 1 or 2 oxygen, nitrogen and/or sulfur heteroatom(s) .
• This invention further relates to a compound of formula
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen or C ⁇ -C 3 alkyl
• R 9 and R 10 are independently hydrogen, C ⁇ -C 3 alkyl, phenyl or benzyl; and n is 1-3; provided that if A 1 , A 3 and A 5 are independently hydrogen, C ⁇ -C 6 alkyl, C ⁇ -C 6 alkoxy, halo, nitro, phenyl, phenoxy, benzyl, benzyloxy or -COOH, then neither A 2 nor A 4 are -COOH; and provided that if any adjacent two of A 3 , A 4 and A 5 form with the benzene ring a fused 5- or 6-membered carbocyclic or heterocyclic aromatic ring, said heterocyclic aromatic ring containing 1 or 2 oxygen, nitrogen and/or sulfur heteroatom(s) , then A 2 is not -COOH.
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each hydrogen;
• R 9 and R 10 are independently hydrogen, methyl or benzyl.
• any adjacent two of A 1 , A 2 , A 3 , A 4 and A 5 form with the benzene ring a fused 5- or 6-membered carbocyclic or heterocyclic aromatic ring, said heterocyclic aromatic ring containing 1 or 2 oxygen, nitrogen and/or sulfur heteroatom(s) .
• This invention also relates to a compound of formula III
• X and Y are independently -CR 5 R 6 -, -0-, -S- or -NR- , provided that at least one of X and Y is/are - CR 5 R 6 -;
• Y is -0-, -S- or -NR- ;
• R 7 and R 8 are independently hydrogen or methyl .
• Y is -CR 5 R 6 -;
• a 1 , A 2 , A 3 and A 4 are each hydrogen; and
• a 5 is phenoxy, benzyloxy, aryl, heteroaryl, carbocycle or heterocycle, wherein said phenoxy and benzyloxy are substituted with -COOH, and said aryl, heteroaryl, carbocycle and heterocycle are substituted with one or more substituent (s) selected from the group consisting of cyano and -COOH.
• Possible substituents of said alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle, heterocycle, alkoxy, alkenyloxy, phenoxy, benzyloxy, and fused ring include, without limitation, C ⁇ -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C ⁇ -C 6 alkoxy, C 2 -C 6 alkenyloxy, phenoxy, benzyloxy, hydroxy, carboxy, hydroperoxy, carbamido, carbamoyl, carbamyl, carbonyl, carbozoyl, amino, hydroxyamino, formamido, formyl, guanyl, cyano, cyanoamino, isocyano, isocyanato, diazo, azido, hydrazino, triazano, nitrilo, nitro, nitroso, isonitroso, nitrosamino, im
• carbocyclic and heterocyclic moieties include, without limitation, phenyl, benzyl, naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl, indolyl, isoindolyl, indolinyl, benzofuranyl , benzothiophenyl , indazolyl, benzimidazolyl , benzthiazolyl, tetrahydrofuranyl , tetrahydropyranyl, pyridyl, pyrrolyl, pyrrolidinyl, pyridinyl, pyrimidinyl, purinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinolizinyl, furyl, thiophenyl, imidazolyl, oxazolyl, benzoxazolyl , thiazolyl, isoxazolyl,
• the inventive compounds possess one or more asymmetric carbon center (s) and are thus capable of existing in the form of optical isomers as well as in the form of racemic or non- racemic mixtures of optical isomers.
• the optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes well known in the art, for example by formation of diastereoisomeric salts by treatment with an optically active acid or base and then separation of the mixture of diastereoisomers by crystallization followed by liberation of the optically active bases from these salts.
• useful acids include tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acids.
• a different process for separating optical isomers involves the use of a chiral chromatography column optimally chosen to maximize the separation of the enantiomers.
• Still another available method involves synthesis of covalent diastereoisomeric molecules, for example, esters, amides, acetals, ketals, and the like, by reacting compounds used in the inventive methods and pharmaceutical compositions with an optically active acid in an activated form, an optically active diol or an optically active isocyanate.
• the synthesized diastereoisomers can be separated by conventional means, such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to deliver the enantiomerically pure compound. In some cases, hydrolysis to the parent optically active drug prior to dosing the patient is unnecessary since the compound can behave as a prodrug.
• the optically active compounds of the invention can likewise be obtained by utilizing optically active starting materials.
• This invention relates to a method for inhibiting
• NAALADase enzyme activity in an animal or a mammal comprising administering to said animal or mammal an effective amount of a compound of the invention, as defined above .
• This invention further relates to a method for treating a glutamate abnormality in an animal or a mammal, comprising administering to said animal or mammal an effective amount of a compound of the invention, as defined above.
• Glutamate abnormalities to be treated may be selected from the group consisting of compulsive disorder, stroke, ischemia, demyelinating disease, Parkinson's disease, ALS, Huntington' s disease, schizophrenia, pain, anxiety, anxiety disorder, memory impairment and glaucoma.
• the compulsive disorder is alcohol, nicotine or cocaine dependence .
• Stroke patients often experience a significant temporal delay between the onset of ischemia and the initiation of therapy.
• the inventive compounds have a therapeutic window of opportunity of at least 1 hour. Accordingly, when the glutamate abnormality is stroke, the compound of the invention may be administered to said animal or mammal for up to 60 minutes, 120 minutes or more following onset of stroke .
• preferred compounds of the invention are expected to be those that block glutamate release pre-synaptically without interacting with post-synaptic glutamate receptors. Such compounds would be devoid of the behavioral toxicities associated with post-synaptic glutamate antagonists.
• This invention further relates to a method for effecting a neuronal activity in an animal or a mammal, comprising administering to said animal or mammal an effective amount of a compound of the invention, as defined above .
• the neuronal activity that is effected by the inventive method may be stimulation of damaged neurons, promotion of neuronal regeneration, prevention of * neurodegeneration or treatment of a neurological disorder.
• neurological disorders that are treatable by the inventive methods include without limitation: trigeminal neuralgia; glossopharyngeal neuralgia; Bell's Palsy; myasthenia gravis; muscular dystrophy; ALS; progressive muscular atrophy; progressive bulbar inherited muscular atrophy; herniated, ruptured or prolapsed invertebrate disk syndromes; cervical spondylosis; plexus disorders; thoracic outlet destruction syndromes; peripheral neuropathies such as those caused by lead, dapsone, ticks, porphyria, or Guillain-Barre syndrome; diabetic neuropathy; pain; Alzheimer's disease; and Parkinson's disease.
• the inventive method is particularly useful for treating a neurological disorder selected from the group consisting of peripheral neuropathy caused by physical injury or disease state, diabetic neuropathy, pain, traumatic brain injury, physical damage to spinal cord, stroke associated with brain damage, demyelinating disease and neurological disorder relating to neurodegeneration.
• the compound of the invention is preferably administered in combination with an effective amount of morphine.
• Examples of neurological disorders relating to neurodegeneration include Alzheimer's disease, Parkinson's disease, and ALS.
• This invention further relates to a method for treating a prostate disease in an animal or a mammal, comprising administering to said animal or mammal an effective amount of a compound of the invention, as defined above.
• This invention further relates to a method for treating cancer in an animal or a mammal, comprising administering to said animal or mammal an effective amount of a compound of the invention, as defined above.
• Preferred cancers to be treated are those in tissues where NAALADase resides, including without limitation the brain, kidney and testis.
• This invention further relates to a method for inhibiting angiogenesis in an animal or a mammal, comprising administering to said animal or mammal an effective amount of a compound of the invention, as defined above.
• Angiogenesis may be necessary for fertility or metastasis of cancer tumors, or may be related to an angiogenic-dependent disease.
• inventive methods may also be useful for treating an angiogenic-dependent disease including, without limitation, rheumatoid arthritis, cardiovascular diseases, neovascular diseases of the eye, peripheral vascular disorders, dermatologic ulcers and cancerous tumor growth, invasion or metastasis.
• This invention further relates to a method for
• TGF- ⁇ activity in an animal or a mammal, comprising administering to said animal or mammal an effective amount of a compound of the invention, as defined above .
• Said effecting a TGF- ⁇ activity includes increasing,
• TGF- ⁇ abnormalities examples include neurodegenerative disorders, extra-cellular matrix formation disorders, cell-growth related diseases, infectious diseases, immune related diseases, epithelial tissue scarring, collagen vascular diseases, fibroproliferative disorders, connective tissue disorders, inflammation, inflammatory diseases, respiratory distress syndrome, infertility and diabetes.
• Typical neurodegenerative disorders to be treated include neural tissue damage resulting from ischemia reperfusion injury, myelination and neurodegeneration.
• Typical cell-growth related disorders to be treated include those affecting kidney cells, hematopoietic cells, lymphocytes, epithelial cells and endothelial cells.
• Typical infectious diseases to be treated include those caused by a macrophage pathogen, particularly a macrophage pathogen selected from the group consisting of bacteria, yeast, fungi, viruses, protozoa, Trypanosoma cruzi , Histoplasma capsulatum, Candida albicans, Candida parapsilosis, Cryptococcus neoformans, Salmonella, Pneumocystis, Toxoplasma, Listeria, Mycobacteria, Rickettsia and Leishmania.
• Mycobacteria include without limitation Mycobacterium tuberculosis and Mycobacterium leprae .
• Toxoplasma includes without limitation Toxoplasma gondii .
• Rickettsia includes without limitation R . prowazekii , R . coronii and R . tsutsugamushi .
• infectious diseases to be treated include single or multiple cutaneous lesions, mucosal disease, Chagas' disease, acquired immunodeficiency syndrome (AIDS) , toxoplasmosis, leishmaniasis, trypanosomiasis, shistosomiasis, cryptosporidiosis, Mycobacterium avium infections, Pneumocystis carinii pneumonia and leprosy.
• AIDS acquired immunodeficiency syndrome
• Typical immune related diseases to be treated include autoimmune disorders; impaired immune function; and immunosuppression associated with an infectious disease, particularly, trypanosomal infection, viral infection, human immunosuppression virus, human T cell lymphotropic virus (HTLV-1) , lymphocytic choriomeningitis virus or hepatitis.
• infectious disease particularly, trypanosomal infection, viral infection, human immunosuppression virus, human T cell lymphotropic virus (HTLV-1) , lymphocytic choriomeningitis virus or hepatitis.
• Typical collagen vascular diseases to be treated include progressive systemic sclerosis ("PSS”), polymyositis, scleroderma, dermatomyositis, eosinophilic fascitis, morphea, Raynaud's syndrome, interstitial pulmonary fibrosis, scleroderma and systemic lupus erythematosus .
• PSS progressive systemic sclerosis
• polymyositis scleroderma
• dermatomyositis eosinophilic fascitis
• morphea morphea
• Raynaud's syndrome interstitial pulmonary fibrosis
• scleroderma and systemic lupus erythematosus
• Typical fibroproliterative disorders to be treated include diabetic nephropathy, kidney disease, proliferative vitreoretinopathy, liver cirrhosis, biliary fibrosis, and myelofibrosis.
• Especially preferred kidney diseases include mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic neuropathy, renal interstitial fibrosis, renal fibrosis in transplant patients receiving cyclosporin, and HIV-associated nephropathy.
• Typical connective tissue disorders to be treated include scleroderma, myelofibrosis, and hepatic, intraocular and pulmonary fibrosis.
• Typical inflammatory diseases to be treated are associated with PSS, polymyositis, scleroderma, dermatomyositis, eosinophilic fascitis, morphea, Raynaud's syndrome, interstitial pulmonary fibrosis, scleroderma, systemic lupus erythematosus, diabetic nephropathy, kidney disease, proliferative vitreoretinopathy, liver cirrhosis, biliary fibrosis, myelofibrosis, mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic neuropathy, renal interstitial fibrosis, renal fibrosis in transplant patients receiving cyclosporin, or HIV-associated nephropathy.
• TGF- ⁇ regulating properties include:
• stimulating cell proliferation particularly proliferation of fibroblasts, mesenchymal cells or epithelial cells
• inventive compounds are useful for in vi tro and in vivo diagnostic methods for detecting diseases, disorders and conditions where NAALADase levels are altered including, without limitation, neurological disorders, glutamate abnormalities, diabetic neuropathy, pain, compulsive
• this invention also relates to a method for detecting a disease, disorder or condition where NAALADase levels are altered, comprising: (i) contacting a sample of bodily tissue or fluid with a compound of the invention, as defined above, wherein said compound binds to any NAALADase in said sample; and
• any NAALADase bound to said sample wherein the amount of NAALADase is diagnostic for said disease, disorder or condition.
• bodily tissues and fluids include, without limitation, prostate tissue, ejaculate, seminal vesicle fluid, prostatic fluid, urine, blood, saliva, tears, sweat, lymph and sputum.
• the compound may be labeled with a marker using techniques known in the art.
• Useful markers include, without limitation, enzymatic markers and imaging reagents.
• imaging reagents include radiolabels such as 131 I, ⁇ :L1 In, 123 I, 99 Tc, 32 P, 125 I, 3 H and 14 C; fluorescent labels such as fluorescein and rhodamine; and chemiluminescers such as luciferin.
• radiolabels such as 131 I, ⁇ :L1 In, 123 I, 99 Tc, 32 P, 125 I, 3 H and 14 C
• fluorescent labels such as fluorescein and rhodamine
• chemiluminescers such as luciferin.
• the amount of NAALADase can be measured using techniques known in the art including, without limitation, assays (such as immunometric, calorimetric, densitometric, spectrographic and chromatographic assays) and imaging techniques (such as magnetic resonance spectroscopy ("MRS”), magnetic resonance imaging ("MRI”), single-photon emission computed tomography (“SPECT”) and positron emission tomography (“PET”)).
• assays such as immunometric, calorimetric, densitometric, spectrographic and chromatographic assays
• imaging techniques such as magnetic resonance spectroscopy (“MRS"), magnetic resonance imaging (“MRI”), single-photon emission computed tomography (“SPECT”) and positron emission tomography (“PET”).
• MRS magnetic resonance spectroscopy
• MRI magnetic resonance imaging
• SPECT single-photon emission computed tomography
• PET positron emission tomography
• This invention further relates to a diagnostic kit for detecting a disease, disorder or condition where NAALADas
• This invention further relates to a method for detecting a disease, disorder or condition where NAALADase levels are altered in an animal or a mammal, comprising: (i) labeling a compound of the invention, as defined above, with an imaging reagent;
• the amount of NAALADase can be measured in vivo using known imaging techniques, as described above.
• inventive compounds are effective NAALADase inhibitors.
• inventive compounds are expected to have the same uses as the NAALADase inhibitors disclosed in the patents, patent applications and publications incorporated by reference.
• This invention also relates to a pharmaceutical
• composition comprising:
• an effective amount of a compound of the invention (i) an effective amount of a compound of the invention; and (ii) a pharmaceutically acceptable carrier.
• the compound of the invention is present in an effective amount for inhibiting NAALADase enzyme activity
• angiogenesis effecting a neuronal activity or TGF- ⁇ activity, or treating a glutamate abnormality, compulsive disorder, prostate disease, cancer or glaucoma in an animal or a mammal .
• inventive compounds and compositions may be administered locally or systemically by any means known to an ordinarily skilled artisan.
• inventive compounds and compositions may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles .
• parenteral as used herein includes subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, intracranial or intraosseous injection and infusion techniques.
• inventive compounds and compositions should readily penetrate the blood-brain barrier . when peripherally administered. Compounds that cannot penetrate the blood-brain barrier can be effectively administered by an intraventricular route or by other methods recognized in the art. See, for example, U.S. Patents Nos. 5,846,565, 5,651,986 and 5,626,862.
• inventive compounds and compositions may be administered by a single dose, multiple discrete doses or continuous infusion.
• Pump means particularly subcutaneous pump means, are preferred for continuous infusion.
• Dose levels on the order of about 0.001 to about 10,000 mg/kg of the active ingredient compound are useful in the treatment of the above conditions, with preferred levels being about 0.1 to about 1,000 mg/kg, and more preferred levels being about 1 to about 100 mg/kg.
• the specific dose level for any particular patient will vary depending upon a variety of factors, including the activity and the possible toxicity of the specific compound employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of excretion; drug combination; the severity of the particular disease being treated; and the form of administration. Typically, in vi tro dosage-effect results provide useful guidance on the proper doses for patient administration. Studies in animal models are also helpful. The considerations for determining the proper dose levels are well known in the art.
• Any administration regimen well known to an ordinarily skilled -artisan for regulating the timing and sequence of drug delivery can be used and repeated as necessary to effect treatment.
• Such regimen may include pretreatment and/or co-administration with additional therapeutic agents.
• inventive compounds and compositions may be used alone or in combination with one or more additional agent (s) for simultaneous, separate or sequential use.
• the additional agent (s) may be any therapeutic agent (s) known to an ordinarily skilled artisan, including without limitation: one or more compound (s) of the invention; steroids, for example, hydrocortisones such as methylprednisolone; anti-inflammatory or anti-immune drugs, such as methotrexate, azathioprine, cyclophosphamide or
• cyclosporin A cyclosporin A
• interferon- ⁇ antibodies, such as anti-CD4
• agents which can reduce the risk of a second ischemic event such as ticlopidine; chemotherapeutic agents; immunotherapeutic compositions; electromagnetic radiosensitizers; and morphine.
• inventive compounds and compositions can be co-
• formulation may contain from about 0.01% to about 99.99%
• inventive compounds can be readily prepared by standard techniques of organic chemistry, utilizing the general synthetic pathways depicted below in SCHEMES I, II, and III.
• Precursor compounds are either commercially available or may be prepared by methods known to a person of skill in the art.
• reaction mixture was stirred at -40 °C for 4 hours.
• 31.8 (3C) , 32.4, 33.3, 35.6, 39.0, 59.5, 67.7, 126.2, 127.7, 128.9, 129.6, 130.7, 131.5, 132.9, 137.8, 146.2, 152.6, 170.1, 174.5.
• benzenepropanoic acid (2.70 g, 10.1 mmol) was divided into multiple samples of equal volume, and each of them was passed through a CHIRAPAK.
• AD column 250 mm x 21 mm id
• carbon dioxide/methanol 77/23, v/v
• 3-H-NAAG was then added to each assay tube and incubated for an additional 15 minutes in a 37EC water bath.
• the assay was stopped by adding 1.0 ml of 0.1 M sodium phosphate.
• Glutamate released by the action of the NAALADase enzyme was separated from the assay solution using an anion exchange resin.
• the resin was equilibrated to 25EC, 2.0 ml of the resin was added to a Pasteur pipette pre-loaded with a single glass bead, and each column was washed twice with distilled
• EXAMPLE 6 In Vitro Assay on Ischemia To examine the in vi tro effect of the inventive compounds on ischemia, cortical cell cultures were treated with various compounds of this invention during an ischemic insult utilizing potassium cyanide and 2-deoxyglucose, and for one hour thereafter. For a description of the experimental method used, see Vornov et al., J " . Neurochem. , Vol. 65, No. 4, pp. 1681-1691 (1995). The results are provided below in TABLE III. Neuroprotective effect is expressed as EC 50 , the concentration of the compound, which is required to cause a 50% reduction in glutamate toxicity following an ischemic insult.
• FIGS. 1 and 2 show increased concentrations of TGF- ⁇ l (FIG.
• TGF- ⁇ ' s promotes the release of endogenous TGF- ⁇ ' s from glial cells, which in turn provides neuroprotection for neighboring neurons .
• TGF- ⁇ neutralizing antibodies were then added to the
• FIG. 3 shows that the TGF- ⁇
• FIG. 4 shows that the addition of another growth factor antibody, FGF antibody, did not block the neuroprotective effect of Compound C.
• FGF antibody another growth factor antibody
• FIG. 6 shows that treatment of MCAO
• NAALADase inhibition provides neuroprotection, at least in part, by
• FIG. 5 shows that TGF- ⁇ neutralizing antibodies significantly attenuated the neuroprotective effect of Compound C in vivo.
• NAALADase inhibitors may have implications not only in stroke, but also in other diseases, disorders and conditions including, without limitation, neurological diseases, psychiatric diseases, demyelinating diseases, prostate cancer, inflammation, diabetes and angiogenesis.
• mice Male Sprague-Dawley rats (200-225 g) were rendered diabetic by intravenous administration of streptozotocin ("STZ", 70 mg/kg in phosphate buffered saline). Diabetic animals were divided into five groups: one group receiving Compound A (10 mg/kg or 1 mg/kg) , Compound D (10 mg/kg or 1 mg/kg) or vehicle. Another group of animals (non-STZ treated) served as non-diabetic controls. Drug/vehicle treatment was started in diabetic animals 45 days post-STZ administration. STZ-induced diabetic rats were tested for sensitivity to a heat source as soon as blood glucose levels rose to 320 mg/dl or above (30 days post STZ) .
• STZ streptozotocin
• the rats were then acclimated to a Hargreaves apparatus and thermal nociception was monitored using an infrared heat source directed into the dorsal surface of the hindpaw, and the time taken for the animal to remove its paw noted to the nearest 0.1 seconds (see Hargreaves et al . , supra, for detailed experimental method) .
• the intensity of the beam source was adjusted such that the mean latency for control animals (non- STZ treated) was approximately 10 seconds.
• Each animal was tested 8 times and the mean difference score (between mean non-diabetic control latency and mean diabetic latency) are graphically presented in FIGS. 7A and 7B.
• Diabetic rats displayed a hyperalgesia (shorter response latency) compared to non-diabetic controls, starting 30 days post STZ treatment and progressively worsening in vehicle treated rats. This hyperalgesic response was completely reversed in diabetic rats receiving treatment with Compound D or A (10 mg/kg i.p. daily) . Thus, the results show that NAALADase inhibition attenuates neuropathic pain.
• rats Following sciatic nerve ligation, the rats
• the difference scores are graphically presented in FIG. 8. The results show that NAALADase inhibition attenuates CCI-associated hyperalgesia.
• results are graphically presented in FIG. 11.
• the results show that diabetic rats displayed a hyperalgesia (shorter response latency) compared to non-diabetic controls.
• Diabetic drug-treated rats both Compound D and Compound A displayed longer withdrawal latencies than diabetic vehicle- treated rats, starting after 4 weeks of treatment and persisting through the six months of treatment .
• Nerve conduction velocity was also measured every two weeks through the first eight weeks of treatment and every month thereafter through to the six months of treatment (see De Koning et al . , Peptides, Vol. 8, No. 3, pp. 415-22 (1987) for a description of the experimental method) .
• the results are graphically presented in FIG. 12.
• Diabetic animals generally showed a reduction in nerve conduction velocity compared to non-diabetic controls.
• diabetic animals receiving daily injections of NAALADase inhibitor either Compound D or Compound A at a dose of 10 mg/kg
• Diabetic vehicles on the other hand, showed a progressive deterioration in nerve conduction velocity from 6 to 16 weeks after start of vehicle administration which was maintained through to six months .
• NAALADase inhibition alters the progression of diabetic neuropathy.
• Rats (200 to 225 g) were injected with STZ (70 mg/kg) into the tail vein. Diabetes (>350 mg/dl) was confirmed in all rats, 4 weeks after STZ administration. Rats were left untreated until 35 to 49 days after STZ. Compound D (1, 3, or 10 mg/kg) , Compound E (10 mg/kg) , or vehicle were dosed daily p.o. following confirmation of hyperalgesia and/or nerve conduction velocity deficits. In separate experiments, onset of treatment was delayed until 60 to 90 days- after STZ administration. Nerve conduction velocity or withdrawal response to thermal stimulation of hind paws was measured at intervals, usually bi-weekly for thermal response and monthly for nerve conduction velocity.
• mice Spontaneously diabetic mice (db/db mice) and non- diabetic littermates were obtained from Jackson labs. Mice were left untreated until 7 to 8 months of age (or after 4 to 5 months of chronic diabetes) and then dosed daily with Compound F (1 mg/kg) p.o. Nerve conduction velocity was measured prior to the onset and after eight weeks of treatment.
• Animals were acclimated to the apparatus for at least 5 min.
• An infra-red source was placed under below the plantar surface of the rat hind-paw. The intensity of the source was adjusted so that latency for normal rats was about 10 sees.
• Animals were tested for thermal response latency according to the method of Hargreaves et al (Pain 77-88, 1988) . Each animal was tested 8 times (4 each hind limb) and the latency of response recorded automatically to nearest 0.1 sec. An average of the last 4 measurements for each paw was calculated (8 total measurements) and noted for each rat .
• FIG. 31 shows the effect of NAALADase inhibitor
• NAALADase inhibitor treatment (5 weeks post STZ) .
• neuropathic hyperalgesia was reversed towards normal in both NAALADase inhibitor treated groups. This reversal persisted throughout the subsequent hypoalgesic phase usually seen in prolonged diabetic-STZ rats, with a reduced hypoalgesic phase displayed in
• FIG. 32 shows the motor nerve conduction velocity measurements in STZ diabetic rats and non-diabetic controls prior to and at time periods after NAALADase inhibitor treatment.
• both NAALADase inhibitors Compound D and Compound E reversed the motor nerve conduction velocity towards normal (non-diabetic values) . This effect persisted through 12 weeks of treatment .
• FIG. 33 shows sensory nerve conduction velocity deficits, similarly tested.
• NAALADAse inhibitor treatment similarly reversed sensory nerve conduction velocity deficits, significantly so after only 2 weeks of treatment.
• FIG. 34 shows neuropathic pain abnormalities in another experiment where treatment with lower doses (1 and 3 mg/kg) of the NAALADase inhibitor Compound D was initiated after 7 weeks of STZ treatment. Significant reduction in pain abnormalities were again apparent with both doses of Compound D.
• FIGS. 35 and 36 show sensory and motor nerve conduction velocity, respectively, in these chronically diabetic STZ rats treated with the lower doses of Compound D. Sensory nerve conduction was significantly improved towards normal within 4 weeks of treatment whereas motor nerve conduction remained unimproved by these low doses, even 8 weeks after dosing.
• FIGS. 37 and 38 show sensory and motor nerve conduction velocity measurements generated from an external CRO in a similar chronically diabetic STZ model, where rats were left untreated until 60 days after STZ treatment. Partial reversal of both deficits was again produced by Compound D treatment.
• FIG. 39 shows the same where treatment was delayed yet further, until 90 days after STZ.
• FIG. 40 shows nerve conduction velocity measurements from a genetic mouse model of diabetes, at 6 to 7 months of age (after about 4 months of chronic diabetes) . A significant impairment in sensory NCV was apparent at this time.
• FIG. 41 shows the nerve conduction velocity in these mice after 8 weeks of treatment with another, more potent NAALADase inhibitor administered at 1 mg/kg daily. Significant improvement in the sensory nerve conduction was apparent following drug treatment.
• EXAMPLE 14 Effect of NAALADase Inhibitors on Onset of ALS
• FALS familial amyotrophic lateral sclerosis
• One month old transgenic G1H mice were treated with daily intraperitoneal injections of a vehicle (50 mM HEPES-buffered saline) or a NAALADase inhibitor (50 mg/kg Compound A) .
• Clinical symptoms of the mice were monitored daily. The onset of clinical disease was scored by examining each mouse for its shaking of limbs when suspended in the air by its tail, cross spread of spinal reflexes, hindlimb paralysis, body weight and wheel running activity.
• mice treated with a NAALADase inhibitor show that disease onset was delayed in mice treated with a NAALADase inhibitor .
• mice model of FALS The effect of NAALADase inhibitors on ALS survival and clinical symptoms was tested using again the transgenic mice model of FALS .
• One month old transgenic G1H mice were treated daily with a vehicle (50 mM HEPES-buffered saline) or a NAALADase inhibitor (30 mg/kg Compound B) p.o. (by oral administration) .
• Clinical symptoms of the mice were monitored twice a week. Such symptoms included shaking of limbs, gait, dragging of hind limbs, crossing of limbs, righting reflex and mortality. Gait and crossing of limbs were graded on an arbitrary scale ranging from 0 to 3, with 0 representing most normal and 3 representing least normal, e.g. severest difficulty in walking or crossing limbs.
• Righting reflex was measured by the time (seconds) it took the mice to right themselves when placed on their sides on a flat surface .
• the rats were treated daily with intraperitoneal injections of either a vehicle (23 rats with 50 mM HEPES-buffered saline) or a NAALADase inhibitor (11 rats with 10 mg/kg of Compound A and 22 rats with 10 mg/kg of Compound B) .
• 11 saline treated rats, 11 Compound A treated rats and 11 Compound B treated rats were sacrificed at 8 weeks, and the remaining rats at 12 weeks, after initial IOP elevation.
• OPTIC NERVE TRANSECTION The optic nerve was transected unilaterally in 26 rats under intraperitoneal pentobarbital anesthesia. The conjunctiva was opened with scissors and the optic nerve exposed by traction on extraocular muscles.
• the transection was performed with microscissors 5 mm posterior to the globe, with specific attention to avoidance of injury to major ocular blood vessels.
• the retina was examined ophthalmoscopically to assure that the retinal arterial blood supply was not disrupted.
• the conjunctiva was closed with absorbable suture and the eye dressed with antibiotic ointment.
• the rats were treated daily with intraperitoneal injections of either a vehicle (9 rats with 50 mM HEPES-buffered saline) or a NAALADase inhibitor (8 rats with 10 mg/kg of Compound A and 9 rats with 10 mg/kg of Compound B) .
• 5 saline treated rats, 3 Compound A treated rats and 4 Compound B treated rats were sacrificed at 2 weeks, and the remaining rats at 4 weeks, after transection.
• OPTIC NERVE COUNTING The rats were sacrificed by exsanguination under deep pentobarbital anesthesia. They were perfused through the heart with 2% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, and the eyes with attached optic nerves were removed. A cross- section of the optic nerve from both experimental (glaucoma or transection) and control eyes was removed 1.5 mm posterior to the globe, 1 mm in thickness, and post-fixed in 2% osmium tetroxide in buffer. These were processed into epoxy resin, sectioned at 1 micron and stained with toluidine blue.
• the area of the optic nerve cross-section was measured by outlining its outer border at lOx magnification on an image analysis system (Universal Imaging Corp., Westchester, PA) with Synsys digital camera and Metamorph software. Three area measurements were taken and the mean value was determined.
• images were captured- with a lOOx phase contrast objective from 10 different areas of each nerve. These were edited to eliminate non-neural objects and the size of each axon internal to the myelin sheath (its minimum diameter) and the density of axons/square mm were calculated for each image and nerve. The mean density was multiplied by total nerve area to yield fiber number for each nerve.
• the total fiber number in glaucoma or transection nerves was compared to the normal, fellow eye of each rat to yield a percent loss value.
• the number of axons counted among the 10 images was an approximately 20% sample of the 80-90,000 axons in normal rat nerves .
• the person measuring axon number was masked to the protocol conducted on the nerves.
• OP Integral Difference difference in IOP exposure between glaucoma eye and normal eye in each rat (mm Hg -- days) .
• Percent Difference mean percent difference in fiber number between glaucoma and normal eye in each rat (positive value indicates fewer fibers in the glaucoma eye) .
• Percent Difference mean percent difference in fiber number between glaucoma and normal eye in each rat (positive value indicates fewer fibers in the glaucoma eye) .
• Transgenic HD mice of the N171-82Q strain and non- transgenic littermates were treated with NAALADase inhibitor Compound B (30 mg/kg) or a vehicle from 10 weeks of age.
• the mice were placed on a rotating rod ("rotarod") .
• the length of time at which the mouse fell off the rotarod was recorded as a measure of motor coordination.
• FIG. 42 shows that transgenic HD mice treated with Compound B stayed longer on the rotarod than similar transgenic HD mice treated with a vehicle.
• the treatment with Compound B had no effect on the rotarod performance of normal non-HD mice .
• FIG. 43 shows that while the vehicle treated HD mice demonstrated the lowest mean locomotor score, the treatment with NAALADase inhibitor had no apparent effect on overall locomotion.
• mice Thirteen mice (six male and seven female) were assigned
• FIG. 44 shows the survival distributions over time
• the median survival time is 184
• FIGS. 45 and 46 show the survival distributions
• EXAMPLE 18 A patient is suffering from any disease, disorder or condition where NAALADase levels are altered, including any of the diseases, disorders or conditions described above. The patient may then be administered an effective amount of a compound of the invention. It is expected that after such treatment, the patient would not suffer any significant injury due to, would be protected from further injury due to, or would recover from the disease, disorder or condition. All publications, patents and patent applications identified above are herein incorporated by reference, as though set forth herein in full.

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